• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
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    • 专利摘要:
    The present invention relates to a composition for storing at least one biomolecule, the composition comprising: a. Between about 0.2 weight percent and about 20.0 weight percent D-5 (+) - trehalose dehydrate; and B. between about 0.1 weight percent and about 10.0 weight percent D-mannitol; c. between about 0.01 weight percent and 0.3 weight percent polyoxyethylene sorbitan monooleate; and d. between about 1.0 nM and about 500 mM sodium citrate with a pH between about 4.0 and about 8.0; and e. between about 0 weight percent and about 0.5 weight percent preservative solution, said preservative solution comprising: i. between about 1.0 weight percent and about 5.0 weight percent 5-chloro-2-methyl-4-isothiazolin-3-one; and ii. between about 0.1 weight percent and about 3.0 weight percent 2-methyl-4-isothiazolin-3-one.
    公开号:BE1023343B1
    申请号:E2015/5418
    申请日:2015-07-01
    公开日:2017-02-09
    发明作者:Didier Falconnet；Lucienne Lagopoulos
    申请人:Mycartis Nv；
    IPC主号:
    专利说明:

    STORAGE BUFFER
    Description
    The present invention relates to a composition for storing a biomolecule and uses thereof to store the biomolecule in a liquid phase. The invention also relates to a method for storing a biomolecule in a solid phase and a method for storing a biomolecule in a liquid phase.
    Biomolecules can be in an active or inactive state. The active state of a biomolecule is based on covalent and non-covalent interactions between atoms that form the three-dimensional structure of the biomolecule and thus guarantee a functional and active state of the biomolecule. On the other hand, when the biomolecule is stored under unsuitable conditions, at least a portion of the non-covalent interactions are altered and the biomolecule becomes inactive. For example, enzymes in the active state are able to catalyze biochemical reactions, while no biochemical reactions can be catalyzed if the enzymes are in an inactive state.
    To perform a biological analysis, it was usually necessary that commercially available biomolecules were supplied by the supplier in a suitable storage buffer. In this regard, the purpose of the storage buffer is to keep the biomolecule in active state.
    In fact, biomolecules are stored in a solid phase by lyophilization or in a liquid phase. Lyophilization typically involves consecutive freeze-drying steps applied to a buffered solution of the active biomolecule. Another possibility is to dilute the active biomolecule in a special storage buffer solution for liquid storage. In general, solid or liquid phase storage is typically chosen depending on the type of biomolecule, the stability of the biomolecule and the duration of storage.
    Existing storage buffers are typically specifically designed for storage in the liquid phase or storage in the solid phase. To the applicant's knowledge, there is no storage buffer suitable for multiple purposes for storing biomolecules in solid and liquid phases.
    During the process of optimizing the storage of a product, the applicant is looking for a unique storage buffer to store the biomolecule either in the solid state or in the liquid state.
    The present invention satisfies these objectives by providing a composition for storing at least one biomolecule, the composition comprising: a. Between about 0.2 weight percent and about 20 weight percent D - (+) - trehalose dehydrate; and B. between about 0.1 weight percent and about 10 weight percent D-mannitol; c. between about 0.01% by weight and 0.3% by weight of polyoxyethylene sorbitan monooleate; and d. between about 1.0 nM and about 500 mM sodium citrate with a pH between about 4.0 and about 8.0; and e. between about 0 weight percent and about 0.5 weight percent preservative solution, said preservative solution comprising: i. between about 1.0 weight percent and about 5.0 weight percent 5-chloro-2-methyl-4-isothiazolin-3-one and ii. between about 0.1 weight percent and about 3.0 weight percent of 2-methyl-4-isothiazolin-3-one.
    The invention also relates to a method for storing in liquid phase at least one biomolecule, the method comprising the successive steps of: i. providing a container comprising said biomolecule; ii. dissolving said biomolecule with a composition according to the present invention; iii. filling the container with an inert gas; iv. closing the container.
    The invention further relates to a method for storing at least one biomolecule in the solid state, the method comprising the successive steps of: i. providing a container comprising said biomolecule; ii. dissolving said biomolecule with a composition according to the present invention; iii. lyophilizing said biomolecule by applying at least: 1. a freezing step at a first pressure P1; and 2. a primary drying step at a second pressure P2, wherein said P2 is lower than said first pressure P1; and iv. filling the container with an inert gas; v. closing the container.
    Finally, the invention relates to the use of the composition according to the present invention for storing at least one biomolecule in liquid phase.
    Thus, the present invention solves the problem by providing a composition that makes it possible to store at least one biomolecule in solid or liquid phase. The composition of the present invention provides flexibility with respect to the storage temperature, so that the composition can be used to store biomolecules that require different storage temperatures.
    Biomolecules coupled to a solid support may be susceptible to degradation due to the interaction of the biomolecule with said solid support. However, the composition of the present invention has proven to be efficient for storing biomolecules coupled to a solid support, both in the liquid state and in the solid state. Thus, the composition appears to be suitable for storing proteins that are coupled to microparticles. When it comes to storing biomolecules, the composition of the present invention provides better performance than existing storage buffers, both for solid and liquid storage, so that the proportion of the biomolecule in the active state in said composition remains higher than the proportion for a longer period of time of the biomolecule in active state in existing storage buffers, as illustrated in particular in Example 3 below in a 42-day study at -20 ° C, 4 ° C, 24 ° C and 37 ° C. Therefore, the composition of the present invention further enables the storage of the biomolecule, and more particularly the retention of biomolecules in the active state, both in the liquid state and in the solid state.
    For storage in the solid state, for example by lyophilization by a freeze-drying process, various steps at different pressures and temperatures were usually required. The liquid storage process, on the other hand, comprises a limited number of steps, in which in most cases the storing biomolecule is simply mixed into the liquid storage composition and the air above the liquid is replaced by an inert gas. Advantageously, the composition of the present invention is particularly adapted to store at least one biomolecule in the liquid phase. In particular, the composition according to the present invention makes it possible to store biomolecules in the active state at room temperature, for example between 20 ° C and 30 ° C, which is very interesting in view of storing biomolecules in a laboratory. In that regard, it appears that the presence of saccharide derivatives, for example the trehalose and the mannitol in the composition according to the present invention, play an important role in maintaining the activity of the biomolecule.
    According to an embodiment, the composition according to the invention comprises; a. between about 8.0% by weight and about 10% by weight of D - (+) - trehalose dehydrate; and B. between about 1.8 weight percent and about 2.2 weight percent D-mannitol; and c. between about 0.04% by weight and 0.06% by weight of polyoxyethylene sorbitan monooleate; and d. between about 45 nM and about 55 mM sodium citrate with a pH between about 5.5 and 6.5; and e. between about 0 weight percent and about 0.25 weight percent preservative solution, said preservative solution comprising: i. between about 2.1 weight percent and about 2.5 weight percent of 5-chloro-2-methyl-4-isothiazolin-3-one; and ii. between about 0.6 percent by weight and about 0.8 percent by weight of 2-methyl-4-isothiazolin-3-one. Advantageously, the composition according to this embodiment considerably improves the biomolecule's share in the active state relative to the existing storage solutions. Moreover, said composition also makes it possible to store the biomolecule in the active state.
    In one embodiment, the at least one biomolecule is coupled to a solid support. Advantageously, the composition according to the present invention is suitable for storing biomolecules that are coupled at least to a solid carrier. For example, the present invention is particularly suitable for storing antibodies that are coupled to at least one microparticle.
    In one embodiment, a storage buffer for storing at least one biomolecule in liquid and / or solid storage consists of the composition of the present invention.
    In one embodiment, the biomolecule is a protein, preferably an antibody.
    In one embodiment, the inert gas is selected from nitrogen or argon or a mixture thereof.
    According to the present invention, the term "storing at least one biomolecule" refers to storing a biomolecule after its production in a suitable medium, said medium being suitable to limit the denaturation of the biomolecule.
    According to the present invention, the term "preservative solution" refers to a solution that is capable of limiting the development of bacteria and / or fungi and / or yeast in the composition when mixed with said composition.
    In one embodiment, the term "liquid state" refers to a composition that is substantially in the liquid state, preferably in the liquid state.
    According to the present invention, the term "storing at least one biomolecule in the liquid phase" refers to at least one biomolecule that is stored in a composition that is substantially in the liquid state, preferably in the liquid state.
    In one embodiment, the term "solid state" refers to a composition that is substantially in a solid state, preferably in a solid state. The solid state can be obtained by various methods. Said methods are selected, for example, from freezing, freeze drying or lyophilization.
    According to the present invention, the term "storage of at least one solid phase biomolecule" refers to at least one biomolecule that is stored on a composition that is substantially in the solid state, preferably in the solid state.
    According to the present invention, the term "activity of a protein" refers to the relative proportion of the active state and of the inactive state of said protein. In the active state, the protein is functional and effective to perform its function. For example, if the protein is an enzyme, said enzyme in the active state is able to catalyze biochemical reactions, while no biochemical reactions can be catalyzed when the enzyme is in the inactive state.
    The present invention is further illustrated by the following detailed description set forth in the light of the attached figures, which represent an exemplary and explanatory embodiment of a composition for storing at least one biomolecule, and uses thereof. The description includes the following figures: Figure 1a shows the development of fluorescence during immunological assays aimed at detecting the capture antibody IL-6 in MCBS buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 1b shows the development of fluorescence during immunological assays aimed at detecting the capture antibody TNF-alpha in MCBS buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 1c shows the development of fluorescence during immunological assays aimed at detecting IL-1 beta in MCBS buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 1d shows the development of fluorescence during immunological assays aimed at detecting IL-2 in MCBS buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 2a shows the development of fluorescence during immunological assays aimed at detecting capture antibody IL-6 in PBS buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 2b shows the development of fluorescence during immunological assays aimed at detecting the capture antibody TNF-alpha in PBS buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 2c shows the development of fluorescence during immunological assays aimed at detecting IL-1 beta in PBS buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 2d shows the development of fluorescence during immunological assays aimed at detecting IL-2 in PBS buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 3a shows the development of fluorescence during immunological assays aimed at detecting capture antibody IL-6 in PBSTNa buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 3b shows the development of fluorescence during immunological assays aimed at detecting capture antibody TNF-alpha in PBSTNa buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 3c shows the development of fluorescence during immunological assays aimed at detecting IL-1 beta in PBSTNA buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 3d shows the development of fluorescence during immunological assays aimed at detecting IL-2 in PBSTNa buffer for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 4a shows the development of fluorescence during immunological assays aimed at detecting capture antibody IL-6 in SG02® buffer (StabilGuard Choice Microarray Stabilizer) for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 4b shows the development of fluorescence during immunological assays aimed at detecting the capture antibody TNF-alpha in SG02® buffer (StabilGuard Choice Microarray Stabilizer) for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 4c shows the development of fluorescence during immunological assays aimed at detecting IL-1 beta in SG02® buffer (StabilGuard Choice Microarray Stabilizer) for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C; Figure 4d shows the development of fluorescence during immunological assays aimed at detecting IL-2 in SG02® buffer (StabilGuard Choice Microarray Stabilizer) for 42 days at -20 ° C, 4 ° C, 24 ° C and 37 ° C.
    Example 1: Preparation of a storage buffer
    A storage buffer comprising a composition according to the present invention is prepared by the following steps: 1. preparing a solution 1, comprising: i. 5.25 g citric acid monohydrate (CAS number 5949-29-1) ii. 500 ml of water (CAS number 7732-18-5) 2. preparing a solution 2, comprising: i. 29.41 g of sodium citrate dehydrate (CAS number 6131-04-3) ii. 2000 ml of water (CAS number 7732-18-5) 3. mixing 230 ml of solution 1 and 1770 ml of solution 2 to obtain solution 3; 4. adjusting solution 3 to pH 6 by adding solution 1 and / or solution 2 to solution 3; 5. preparing solution 4 by mixing solution 3 with: i. 1.0 g Tween 80 (CAS number 9005-65-6) ii. 180 g of D - (+) - trehalose dehydrate (CAS number 6138-23-4) iii. 40 g of D-mannitol (CAS number 69-65-8) 6. filtering solution 4 by passing solution 4 through a 0.2 micron filter membrane; 7. preparing solution 5 by mixing solution 4 from step 6 with 2 ml of ProClin® 300 (ProClin® 300 marketed by Sigma-Aldrich, comprising 5-chloro-2-methyl-4-isothiazolin-3 one, CAS number 26172-55-4; and 2-methyl-4-isothiazolin-3-one, CAS number 2682-20-4).
    The storage buffer prepared herein is suitable for storing at least one biomolecule in liquid and solid phase, preferably in liquid phase.
    Example 2: lyophilization method for lyophilization of biomolecules coupled to microparticles
    The lyophilization method comprises the following steps: 1. Providing a container comprising the biomolecules or biomolecules grafted onto microparticles. The container can for example be a test tube or a cassette. In the present case, the container is a test tube comprising approximately 20,000 microparticles; 2. Contacting said microcarriers with the correct amount of a composition according to the present invention prepared according to Example 1 above. At the present CAS numbers, 3.3 microliters of the composition was delivered to the test tube, said solution comprising: * 3% (m / v) trehalose, 2% (m / v) mannitol, 0.05% Tween80, 10 mM sodium citrate buffer with pH 6; 3. Lyophilizing the microcarrier contained in the composition by applying a freeze-drying method comprising: * a freezing step: from room temperature (about 20 ° C) to -50 ° C for 3h50 at ambient pressure (about 1 bar); followed by * a primary freeze-drying step: from -50 ° C to 30 ° C for 22 hours at 40 microbar; 4. Bringing the resulting lyophilized microcarrier in the nitrogen container into contact to store it under a nitrogen atmosphere; 5. Closing the holder.
    Example 3: Comparison of the activity of four proteins in four candidate storage buffers
    The present example is aimed at investigating the activity of proteins coupled to microparticles in four candidate storage buffers.
    For the present study, the candidate storage buffers are: * MCBS: MyCartis Biomolecule Storage Buffer, prepared according to Example 1 mentioned above; * PBS-based buffer (PBS stands for Phosphate Buffered Saline, phosphate buffered saline): i. The PBS-based buffer comprises 10 mM PBS, 0.1% BSA (CAS number 9048-46-8), 0.02% Tween20 (CAS number 9005-64-5), 0.05% sodium azide (CAS- number 26628-22-8); ii. The pH is adjusted to 7.2-7.4 and said PBS-based buffer is filtered on a 0.2 micron filter membrane; * PBSTNa buffer: i. The PBSNa buffer comprises 10 mM PBS, 0.3% Tween20 (CAS number 9005-64-5), 0.05% sodium azide (CAS number 26628-22-8); ii. The pH is adjusted to 7.2-7.4 and said PBSNa buffer is filtered on a 0.2 micron filter membrane. * SG02®: StabilGuard Choice Microarray Stabilizer (SG02® marketed by SurModics, Eden Prairie, Minnesota, USA).
    In the present study, two capture antibodies IL-6 (clone MQ2-13A5) and TNF (clone MAb1, marketed by BD Biosciences®) and two cytokines (IL-1 beta and IL-2) were coupled to the microparticles. Prior to the study, the applicant has checked whether the four proteins can be tested in a multiplex assay format without significant cross-reactivity / non-specific signal.
    To determine the proportion of the active state of each of the proteins that are of interest in each of the candidate storage buffers, the following 7-step procedure was performed: 1. linking one type of protein of interest (selected from capture antibodies IL-6 or TNF and cytokines IL-1 beta and IL-2 in the present example) on a set of microparticles, each set of microparticles being coded with a specific code to identify the proteins coupled thereto; 2. splitting each set of microparticles into four equal parts, and mixing a part in MCBP, a part in PBS-based buffer, a part in PBSTNa and a part in SG02®; 6. Determining the proportion of active protein by measuring the fluorescence of each series of microparticles on each test day in functional multiplex assays; in the present study, the measured fluorescence is emitted by labeled secondary antibodies that are designed to measure the protein of interest that is linked to the microparticle. 7. Graphing the fluorescence of each test day as a function of the storage time and temperature and normalizing the fluorescence with a reference amount stored in each candidate storage buffer at -80 ° C.
    In the present example, the two capture antibodies (IL-6 and TNF) and two cytokines (IL-1 beta and IL-2) were stored at different temperatures in the candidate buffers and tested in multiplex assays on days 1, 7, 14, 28 and 42.
    Each storage temperature has a specific purpose: samples stored at -80 ° C are used as a reference on the assumption that there is no degradation and that variations between the assays are taken into account; results of -80 ° C are used as the normalization reference for the fluorescence on each test day; -20 ° C and 4 ° C are convenient storage temperatures that are used to determine the proportion of the active state in real time; 24 ° C and 37 ° C have the goal of accelerated aging and allow early evaluation of buffer candidates.
    In the present example, the fluorescence was measured at -80 ° C, -20 ° C, 4 ° C, 24 ° C and 37 ° C. During the immunological assays, said protein can be detected when it is coupled to the microparticles in active state, so that a fluorescence signal is emitted. On the other hand, when in idle state, the protein is not detected and no signal is transmitted. Therefore, the measured fluorescence makes it possible to estimate the relative proportions of biomolecules in active state and inactive state.
    When IL-6 and TNF alpha capture antibodies are stored under extreme conditions, ie 37 ° C for 42 days, losses of 40 to 90% of the active state are observed, except when stored in MCPS buffer, where the loss of active state is limited to -20%. Therefore MCPS does not disrupt the activity of capture antibodies IL-6 and TNF-alpha. MCPS completely preserves the active state of the two antibodies tested, in contrast to the other candidate buffers. When stored at 24 ° C for 42 days, the loss of active state of both capture antibodies IL-6 and TNF-alpha is very limited in MCBS, whereas this is not the case for the other candidate storage buffers. In addition, data at 4 ° C or -20 ° C did not show a significant loss of active state for the capture antibodies after 42 days in MCBP.
    At -20 ° C, 4 ° C or 24 ° C, no loss of active state is observed for the capture antibodies in MCBP, which is not the case in the other candidate storage buffers. In contrast to other candidate storage buffers, MCBP thus allows the storage of capture antibodies in the active state, in the liquid state or in the solid state. These findings are demonstrated in the present study for storage of capture antibodies in the liquid phase at 4 ° C, 24 ° C and 37 ° C and for solid-state storage at -80 ° C and -20 ° C.
    With regard to cytokines, the loss of active state for IL-2 in MCBP is limited to 50%, which is not the case in the other candidate storage buffers. The data also indicate that at 37 ° C and 24 ° C, the proportion of IL1 beta in active state is more important in MCBS compared to the proportion of IL1 beta in active state in the other candidate storage buffers. If the coupled cytokines are stored in MCPS buffer at 4 ° C or lower, no significant loss of active state is observed for both cytokines after 42 days, unlike the other candidate buffers.
    Other embodiments of the invention will be apparent to those skilled in the art upon consideration of the description and practice of the invention described in this application. The description and examples are intended to be considered as examples only, the true scope and spirit of the invention being indicated by the following claims.
    权利要求:
    Claims (5)
    [1]
    Conclusions
    A composition for storing at least one biomolecule, the composition comprising: a. Between about 0.2% by weight and about 20.0% by weight of D - (+) - trehalose dehydrate; and B. between about 0.1 weight percent and about 10.0 weight percent D-mannitol; c. between about 0.01% by weight and 0.3% by weight of polyoxyethylene sorbitan monooleate; and d. between about 1.0 nM and about 500 mM sodium citrate with a pH between about 4.0 and about 8.0; and e. between about 0 weight percent and about 0.5 weight percent preservative solution, said preservative solution comprising: i. between about 1.0 weight percent and about 5.0 weight percent of 5-chloro-2-methyl-4-isothiazoline-3-one; and ii. between about 0.1 weight percent and about 3.0 weight percent of 2-methyl-4-isothiazolin-3-one.
    [2]
    The composition of claim 1, comprising: a. Between about 8.0% by weight and about 10.0% by weight of D - (+) - trehalose dehydrate; and B. between about 1.8 weight percent and about 2.2 weight percent D-mannitol; and c. between about 0.04% by weight and 0.06% by weight of polyoxyethylene sorbitan monooleate; and d. between about 45 nM and about 55 mM sodium citrate with a pH between about 5.5 and about 6.5; and e. between about 0 weight percent and about 0.25 weight percent preservative solution, said preservative solution comprising: i. between about 2.1 weight percent and about 2.5 weight percent of 5-chloro-2-methyl-4-isothiazolin-3-one; and ii. between about 0.6 percent by weight and about 0.8 percent by weight of 2-methyl-4-isothiazolin-3-one.
    [3]
    A method for storing at least one biomolecule in a liquid phase, the method comprising the successive steps of: i. providing a container comprising said biomolecule; ii. dissolving said biomolecule with a composition according to claim 1 or 2; iii. filling the container with an inert gas; iv. closing the container.
    [4]
    A method for storing at least one biomolecule in a solid phase, the method comprising the successive steps of: i. providing a container comprising said biomolecule; ii. dissolving said biomolecule with a composition according to claim 1 or 2; iii. lyophilizing said biomolecule by applying at least: 1. a freezing step at a first pressure P1; and 2. a primary drying step at a second pressure P2, wherein said P2 is lower than said first pressure P1; and iv. filling the container with an inert gas; v. closing the container.
    [5]
    Use of the composition according to claim 1 or 2 for storing at least one biomolecule in liquid phase.
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    同族专利:
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    WO2016185011A1|2016-11-24|
    AU2016265442A1|2017-11-23|
    BE1023343A1|2017-02-09|
    EP3297667A1|2018-03-28|
    CN107690438A|2018-02-13|
    JP2018525321A|2018-09-06|
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    法律状态:
    2020-04-16| MM| Lapsed because of non-payment of the annual fee|Effective date: 20190731 |
    优先权:
    申请号 | 申请日 | 专利标题
    EP15168524|2015-05-20|
    EP15168524.5|2015-05-20|
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